The present invention relates to a novel process for isolating superoxide dismutase from red blood cells and more particularly to a process for isolating superoxide dismutase from hemolyzed red blood cells by causing proteins having affinity for hemoglobin and superoxide dismutase and other unnecessary proteins to precipitate from the hemolyzed red blood cells in the presence of monovalent inorganic neutral salts and transition metal salts by the application of heat.
Superoxide dismutase is an enzyme which catalyses disproportionation of superoxide O.sub.2.sup.- ions as follows: EQU 2O.sub.2.sup.- +2H.sup.+ .fwdarw.O.sub.2 +H.sub.2 O.sub.2
This enzyme is widely distributed in animal and plant organisms and has the function of eliminating active oxygen from those organisms, which active oxygen is generated in the course of biochemical reactions in the organisms and works as a cytotoxin in the organisms.
Superoxide dismutase obtained in its pure state from the red blood cells of higher animals is a protein metal chelate, which is named "orgotein."
Bovine erythrocyte superoxide dismutase has a molecular weight of 31,200 and consists of two apparently identical sub-units of the molecule, each sub-unit having 151 amino acids, the greater part of the amino acids having a .beta.-type cylindrical structure and the remainder having an .alpha.-type spiral structure.
Due to the structures of the amino acids, bovine erythrocyte superoxide dismutase is very resistant to denaturation.
As mentioned above, bovine erythrocyte superoxide dismutase is a protein metal chelate, in which copper and zinc are each contained in an amount of 2 gram atoms per mole of the superoxide dismutase.
The isoelectric point of the superoxide dismutase is near pH 5.5.
Superoxide dismutase is innocuous to living organisms and, immunologically, it cannot be regarded as a foreign material in living organisms and therefore it is an injectable protein.
Pharmacologically, superoxide dismutase can serve as a medicine for treatment of inflammation caused by autoimmune diseases, and, recently, by use of superoxide dismutase, there have been attempts to develop medicines for the treatment of arthritis deformans and chronic rheumatism and for treatment of harmful side effects caused by radio therapy.
Conventionally, several methods of isolating erythrocyte superoxide dismutase, namely "orgotein," from red blood cells, have been proposed. However, those conventional methods have a variety of shortcomings. For instance, the purity and yield of the erythrocyte superoxide dismutase obtained are low and preserved blood cannot be employed for producing the erythrocyte superoxide dismutase by those conventional methods. As a matter of fact, if preserved blood could be employed for producing erythrocyte superoxide dismutase in practice, it would be extremely useful.
More specifically, in Japanese Patent Publication Ser. No. 53-31206, there is disclosed a method of isolating orgotein from hemolyzed red blood cells by subjecting the hemolyzed red blood cells to heat treatment in the presence of salts of divalent metals, such as copper, zinc, cobalt, manganese and magnesium, and causing hemoglobin and carbonic anhydrase to precipitate and removing the same.
In this method, although the hemolyzed red blood cells are treated at a comparatively high temperature for a long period of time, separation by precipitation of unnecessary proteins from the hemolyzed red blood cells for isolation of superoxide dismutase is incomplete and red colored hemoglobin remains in the supernatant solution of the heat-treated hemolyzed red blood cells. The unseparated unnecessary proteins cause degrading of the activity of superoxide dismutase during the next purification process of superoxide dismutase. Furthermore, in this method, when lyophilized red blood cells are employed for isolating superoxide dismutase therefrom, separation and purification of superoxide dismutase are more difficult in comparison with the case where untreated fresh red blood cells are employed.
In Japanese Patent Publication Ser. No. 45-39832, there is disclosed a method of isolating orgotein by treating hemolyzed red blood cells with an organic chlorine compound to form a complex compound of hemoglobin and removing hemoglobin in complex-compound form from the hemolyzed red blood cells. In this method, however, a significant amount of superoxide dismutase is attracted to the complex compound and the yield of superoxide dismutase in the supernatant solution of the hemolyzed red blood cells is decreased by more than 20%.
In the Journal of Biochemical Chemistry, Vol. 224, 6051, there is disclosed another method of isolating superoxide dismutase from hemolyzed red blood cells by use of a chlorine compound. In this method, however, the activity of superoxide dismutase is decreased by at least 20% in the first extraction step, in comparision with the activity of unisolated superoxide dismutase.
These superoxide isolation methods using organic chlorine compounds require a large quantity of organic solvent and therefore are not practical in terms of cost and separation efficiency. Furthermore, when lyophilized blood cells are employed, unnecessary proteins including hemoglobin, to which superoxide dismutase is attracted, remain in the supernatant solution of the hemolyzed red blood cells. Therefore, isolation of superoxide dismutase from tne supernatant solution and purification thereof are extremely difficult.
In Japanese Laid-open Patent Application Ser. No. 49-50195, there is disclosed a further method of isolating superoxide dismutase from hemolyzed red blood cells, which method comprises the steps of subjecting the hemolyzed red blood cells to heat treatment; processing the supernatant solution of the heat-treated hemolyzed red blood cells with a proteolytic enzyme; and filtering the processed supernatant solution and separating and purifying superoxide dismutase from the solution by column chromatography and gel-filtration. The shortcomings of this method are that the steps involved in the process using the proteolytic enzyme are complicated and, when old blood cells or lyophilized blood cells are used for producing superoxide dismutase, superoxide dismutase is tightly bonded to unnecessary proteins contained in the blood cells and therefore purification of superoxide dismutase is extremely difficult.
Finally, in Japanese Patent Publication Ser. No. 53-22137, there is disclosed a method of isolating superoxide dismutase from hemolyzed red blood cells by allowing superoxide dismutase contained in hemolyzed red blood cells to be directly attracted to a weak basic ion-exchange resin. In terms of cost and efficiency, this method, however, is not suitable for separation of such a protein as superoxide dismutase contained in an amount ranging from 0.3 to 0.5 weight percent from other proteins. As a matter of fact, this method cannot be employed for producing a large amount of superoxide dismutase from hemolyzed blood cells.
As described above, those conventional superoxide dismutase isolation methods have a variety of shortcomings and cannot be used in practice.